A long-term objective of the proposed study is to identify and understand factors that affect the cleavability of human amyloid precursor protein (APP). The proteolytic cleavage of APP by the beta-secretase is the required first reaction leading to the generation of amyloid beta-peptide, precipitation of which in the brain tissue is widely believed to cause neurodegeneration in Alzheimer's disease. Our preliminary studies indicated that the cleavability of APP was influenced by the protein structure of APP: the removal of APP ectodomain, either by deletion mutagenesis or by protein unfolding, could release the inhibition imposed on the cleavage site by the native protein; familial Alzheimer's disease mutations affected this autoinhibitory mechanism and caused increased cleavage. Since heparin binding to APP reversed the effect of the mutation, it was hypothesized that intracellular heparan sulfates, by forming complexes with APP inside the cell, could function to inhibit the proteolytic reaction. Specific aims of this proposal include: (i) crystallographic characterization of the juxtamembrane domain of APP, which contains the secretase cleavage site; (ii) crystallographic characterization of APP:heparin complexes; and (iii) mutagenesis studies to probe the interaction between heparin and APP, and to investigate the role of intracellular heparan sulfates in the proteolytic processing of APP. The eventual goal is to use this knowledge to help design heparin-like compounds to reinforce the autoinhibitory mechanism for controlling the synthesis of amyloid beta-peptide in aged individuals to prevent Alzheimer's disease.